RT Journal Article
SR Electronic
T1 Designed active-site library reveals thousands of functional GFP variants
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.10.11.511732
DO 10.1101/2022.10.11.511732
A1 Jonathan Yaacov Weinstein
A1 Carlos Martí-Gómez
A1 Rosalie Lipsh-Sokolik
A1 Shlomo Yakir Hoch
A1 Demian Liebermann
A1 Reinat Nevo
A1 Haim Weissman
A1 Ekaterina Petrovich-Kopitman
A1 David Margulies
A1 Dmitry Ivankov
A1 David McCandlish
A1 Sarel Jacob Fleishman
YR 2023
UL http://biorxiv.org/content/early/2023/01/19/2022.10.11.511732.abstract
AB Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is extremely sensitive to mutations due to a high density of molecular interactions, drastically reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called htFuncLib, to design a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We applied htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recovered >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be useful for one-shot optimization of activity in enzymes, binders, and other proteins.Competing Interest StatementThe authors have declared no competing interest.